Volume 111, Issue 6, Pages 905-918 (December 2002) Identification of a Signaling Network in Lateral Nucleus of Amygdala Important for Inhibiting Memory Specifically Related to Learned Fear  Gleb P. Shumyatsky, Evgeny Tsvetkov, Gaël Malleret, Svetlana Vronskaya, Michael Hatton, Lori Hampton, James F. Battey, Catherine Dulac, Eric R. Kandel, Vadim Y. Bolshakov  Cell  Volume 111, Issue 6, Pages 905-918 (December 2002) DOI: 10.1016/S0092-8674(02)01116-9

Figure 1 Strategy for Isolating Amygdala-Enriched Genes (A) Scheme of the differential screening of single cell cDNA libraries from amygdala neurons (with a representative neuron after acute dissociation of the rat amygdala). (B) Op18/Stathmin RNA in situ hybridization on a coronal section of mouse brain. Insets show strong expression in the lateral nucleus of the amygdala and weak expression in the hippocampus. Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)

Figure 2 The Grp Gene Is Specifically Expressed in the Lateral Nucleus/AB of the Amygdala and in the Cued and Contextual CS Pathways to the Amygdala (A) In situ hybridization of the Grp gene in the lateral nucleus of the amygdala and AB with sense (left) and antisense (right) RNA probes. (B1) Mouse brain showing the location of coronal sections C1 and C2. (B2) Major areas that send auditory and contextual information to the amygdala obtained from tract-tracing studies. (C1–2) RNA in situ hybridization shows expression of the Grp gene in the areas shown in B2 diagram. Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)

Figure 3 Expression of the Grpr Gene in the Amygdala (A) The Grpr gene is expressed by interneurons. Left image, fluorescent in situ hybridization for Grpr RNA. Middle image, immunohistochemistry for interneuron marker, glutamic acid decarboxylase (GAD). Right image, Grpr and GAD images combined show colocalization of Grpr in a subset of interneurons. White arrow, example of an interneuron positive both for Grpr and GAD. Black and white arrow, a GAD-positive interneuron that does not express Grpr. CPu, caudate putamen; Cx, cortex. (B) Gross anatomy in the amygdala and in the rest of the brain is normal in GRPR knockout mice (Nissl staining). (C) In situ hybridization showing Grpr expression in the amygdala of wild-type mice (left image). Grpr RNA is absent in the amygdala of GRPR knockout mice (right image). Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)

Figure 4 GRP Receptors Are Functionally Expressed in Interneurons of the Lateral Nucleus of the Amygdala (A1) Bath application of GRP (200 nM) increased frequency of sIPSCs in a pyramidal cell from a control mouse. The effect was blocked by 3 μM bombesin antagonist (n = 6), thus suggesting that the GRP-induced enhancement of GABAergic tonic inhibition was specifically linked to the activation of the GRP receptors. (A2) Effect of GRP on the frequency of sIPSCs is TTX-sensitive, and thus is dependent on action potential firing in interneurons. (A3) GRP failed to increase the frequency of the picrotoxin-sensitive sIPSCs in GRPR knockout mice. (B1) Representative sIPSCs recorded in a pyramidal cell from a control mouse at a holding potential of −70 mV under baseline conditions (left), during GRP application (center), and after the GRPR antagonist was added (right). (B2) Representative sIPSCs recorded in a pyramidal neuron from GRPR knockout mouse under baseline conditions (left), during GRP application (center), and after picrotoxin was added (right). (C) Cumulative amplitude histograms of sIPSCs recorded under baseline conditions (filled symbols) and after GRP was applied (open symbols) in slices from control (left) and GRPR knockout mice. Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)

Figure 5 Pairing-Induced LTP Is Enhanced in GRPR Knockout Mice (A) A schematic representation of a brain slice containing the amygdala that shows position of the recording and stimulation pipettes. (B) LTP of whole-cell EPSCs recorded in the lateral amygdala neuron in response to the cortical input stimulation in slices from control (open symbols) or GRPR knockout (filled symbols) mice. For induction of LTP, the lateral amygdala neuron was held at +30 mV, and 80 presynaptic stimuli were delivered at 2 Hz to the external capsule fibers (arrow). (C) Current-voltage plot of the GABAA receptor IPSCs at holding potentials of −110 mV to −10 mV. Reversal potential of the IPSC mediated by the GABAA receptors was −71 mV. Synaptic currents were recorded in the presence of the AMPA receptor antagonist CNQX (20 μM) and NMDA receptor antagonist D-APV (50 μM). Inset shows GABAA receptor IPSCs recorded at holding potentials of −110 mV to −10 mV. Traces are averages of 10 IPSCs recorded at each holding potential. (D) Pairing-induced LTP of whole-cell EPSCs recorded in the lateral amygdala in wild-type mice under control conditions (open symbols) and in the presence of the bombesin antagonist (3 μM, filled symbols). Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)

Figure 6 GRPR-Deficient Mice Have Enhanced and Resistant Long-Term But Not Short-Term Amygdala-Dependent Fear Memory (A1) Contextual fear conditioning. Significant difference in freezing responses between GRPR knockout mice (n = 9, solid bars) and wild-type (n = 9, open bars) mice was found at 24 hr, 2, 7, and 15 weeks after training. (A2) Cued fear conditioning. In response to the tone (CS), both groups showed an increase in freezing. However, this increase was significantly higher in GRPR knockout animals, although no difference was found between groups in the level of freezing before the onset of the tone (pre-CS). (B1) Contextual and (B2) cued-fear conditioning assessed 30 min or 4 hr after training was normal in GRPR knockout mice. Water maze (C1–4; wild-type, n = 9; knockout, n = 9). In this hippocampus-dependent memory task, both groups of mice showed a similar rate of learning as demonstrating by their equivalent latency (C1) to reach the platform, whether it is during the visible (Day 1 and 2) or hidden platform version of the task (Day 3–6). They displayed the same swimming speed (C2), and thigmotaxis (% of time spent at the periphery; C3). They also showed equivalent performance in the probe trial (% of time spent in the different quadrant areas; C4), which assessed the retention of spatially acquired information necessary to perform this task. GRPR knockout mice are no more sensitive or stressed than wild-type mice (D and E). Pain sensitivity thresholds (D). The intensity of shock required to elicit three reactions, movement (movt), vocalization (vocal), and jump, was assessed and data are presented as the mean ± SEM. No difference was found between groups (wild-type, n = 10; knockout, n = 8). Elevated plus maze assessing basal anxiety (E). No difference was found between GRPR (n = 18) and wild-type mice (n = 16) in the total number of entries, as in the number of entries in the closed or open arms. Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)

Figure 7 A Model for GRP-Dependent Negative Feedback to Principal Neurons in the Amygdala in Wild-Type and GRPR Knockout Mice Cell 2002 111, 905-918DOI: (10.1016/S0092-8674(02)01116-9)